1. Field of the Invention
The present invention is directed to various methods for systematic identification of disease-specific cellular ligands, age-related ligands and receptor-specific ligands as targets for developing ligand-based therapies.
2. Description of the Related Art
Cellular ligands, such as insulin and vascular endothelial growth factor (VEGF), are valuable targets for ligand-based therapies. Ligands like insulin with beneficial roles can be directly used for disease therapy, whereas ligands with detrimental roles, such as VEGF in angiogenic diseases, can be blocked for therapy. Compared to intracellular proteins, cellular ligands have the advantage for convenient extracellular delivery to access and regulate a broad range of receptor-expressing cells. The barrier to developing new ligand-based therapies is conventional approaches to identify unknown cellular ligands on a case-by-case basis with technical challenges. It is even more daunting to reliably predict which ligands may play a role in disease pathogenesis with therapeutic potentials. Therefore, therapeutic ligands are traditionally identified and characterized in individual cases. Ligandomics to systematically identify cell-wide ligands, disease-specific ligands, age-related ligands or receptor-specific ligands for therapy is currently impossible.
Phage display has been widely used to identify cell-binding antibodies or peptides from antibody libraries or random peptide libraries. Identified antibodies or peptides can be used for cell targeting, drug delivery and disease imaging. However, these antibodies or unnatural peptides are not endogenous ligands to delineate disease mechanisms for rational design of novel ligand-based therapies. Similarly, phage display with conventional cDNA libraries of cellular proteins identifies a high percentage of out-of-frame unnatural short peptides due to uncontrollable reading frames of cellular proteins. Thus, conventional phage display cannot be used to efficiently identify cellular ligands. Despite the combination of conventional phage display with next generation DNA sequencing (NGS), this approach is inefficient to identify endogenous ligands owing to the problem of protein reading frame.
To tackle the problem, open reading frame (ORF) phage display has been developed to identify cellular proteins with specific binding or functional activity, including phagocytosis ligands. Other scientists combined OPD next generation DNA sequencing (NGS) to identify protein-protein binding. However, OPD-NGS has not been used for systematic identification of cellular ligands, disease-specific ligands, receptor-specific ligands or age-related ligands.
The challenges to efficiently identify disease-associated or specific cellular ligands by current OPD technology are: a) low efficiency to thoroughly identify enriched ligands by traditional approaches of manually screening phage clones; b) inability to globally quantify the binding or functional activity of all identified ligands; and c) inability to systematically identify disease-specific or age-related ligands. As a result, all disease-associated ligands are traditionally identified on a case-by-case basis with technical challenges. This has hindered reliable selection of cellular ligands as drug targets for rational design of novel ligand-based therapies.